Project Summary/Abstract Maintenance of extracellular potassium (K) in a normal range is essential for the function of cardiac myocytes, skeletal muscle and neurons. Hyperkalemia or hypokalemia is known to cause cardiac arrhythmias and to interfere with normal neuron function and muscle contraction. The aldosterone-sensitive distal nephron (ASDN) including the late distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD) is responsible for K secretion. We previously demonstrated that the depletion Kir.4.1 in the DCT inhibited the expression of NCC which plays an important role in regulating renal K homeostasis. Kir.4.1 is expressed in the DCT, CNT and CCD. Loss-of-function mutations of Kcnj10 cause EAST/SeSAME syndrome in humans (seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalance). The renal phenotype of the disease includes hypomagnesemia, hypokalemia and metabolic alkalosis, suggesting that the disruption of Kir4.1 mainly impairs the transport in the DCT. The role of Kir.4.1 in the regulation of renal K secretion is also strongly suggested by our preliminary data showing that a High K (HK) intake inhibits the basolateral K channels in the DCT while a low K (LK) stimulates the basolateral K channels. We will test two hypotheses: 1) The stimulation of Kir4.1 in the DCT is necessary for inhibiting K secretion during hypokalemia by activating NCC and suppressing ROMK and ENaC thereby switching the function of DCT2 to DCT1; 2) The inhibition of Kir4.1 in the DCT is essential for stimulating K secretion during hyperkalemia by inhibiting NCC and stimulating ROMK and ENaC thereby switching the function of DCT2 to CNT. The hypothesis is based on the following observations made in previous and preliminary experiments: 1) Kir.4.1 is a major type of K channel in the basolateral membrane of the DCT; 2) The disruption of Kir.4.1 abolishes the basolateral K conductance, depolarizes cell membrane and inhibits the Cl conductance in the DCT; 3) The down-regulation of Kir.4.1 decreases NCC expression and activity in inducible kidney specific Kcnj10 knockout mice (KS-Kcnj10 KO); 4) LK intake increases the negativity of the membrane potential (hyperpolarization) in the DCT, indicating a positive correlation between Kir.4.1 and NCC activity; 5) LK-induced stimulation of NCC expression is abolished in KS-Kcnj10 KO; 6) ROMK channel activity in DCT2/CNT of KS-Kcnj10 KO mice is higher than those of WT despite hypokalemia. Therefore, the preliminary data strongly suggest the role of Kir.4.1 in regulating Na and K transport in the DCT in response to dietary K intake. In Aim 1, we will test whether Kir.4.1 activity in the DCT determines NCC expression, phosphorylation and activity via intracellular Cl (Cli)-sensitive mechanism. In Aim 2, we will test whether LK intake-induced stimulation of Kir4.1 is essential for activating NCC and inhibiting ENaC and ROMK in DCT2 thereby suppressing K secretion in the DCT. In Aim 3, we will test whether HK intake-induced inhibition of Kir4.1 is essential for inactivating NCC and stimulating ENaC and ROMK in DCT2 thereby stimulating K secretion in the DCT.